Activation of endothelial cells during inflammatory processes such as atherosclerosis results in dynamic regulation of membrane-anchored adhesion molecules, receptors and ligands that control leukocyte recruitment to sites of injury. One potentially important mechanism for endothelial cells to rapidly modulate their repertoire of cell surface proteins is through proteolytic "shedding". In addition to dynamically altering the cell surface constituents, shedding also leads to the release of soluble ectodomains with distinct biological properties. The ADAM (for A Disintegrin And Metalloproteinase) family of transmembrane proteins are the major ectodomain sheddases identified to date, and we and others have recently demonstrated that a wide variety of proteins involved in leukocyte recruitment and inflammatory responses are `shed'from the cell surface by ADAM17. We have further shown that interference with ADAM17-mediated endothelial or leukocyte shedding significantly inhibits leukocyte transendothelial cell migration in vitro and in vivo, respectively. Although targeted deletion of ADAM17 leads to perinatal lethality, we have developed mice with conditional deletion of ADAM17 that will allow us to eliminate ADAM17 expression from endothelial cells and thus directly test its function in leukocyte recruitment in vivo. This proposal will: 1. further characterize targets and biochemistry of ADAM17-mediated shedding in the endothelium during inflammatory responses;2. Examine the mechanisms involved in transendothelial cell migration dependent upon ADAM17-mediated shedding, and to identify the specific ADAM17 substrates that are contributing to the process;3. Evaluate the impact of targeted deletion of ADAM17 from endothelial cells and leukocytes on inflammatory responses in vivo. The long-term objective of this research is to better understand the cellular and molecular mechanisms controlling chronic inflammatory responses, such as atherosclerosis, that remains the major cause of death in the Western world. PUBLIC HEALTH RELEVANCE: Activation of the endothelial cells that line blood vessels during inflammatory processes such as those associated with cardiovascular disease results in the recruitment of circulating white blood cells into the vessel wall that can ultimately contribute to blockage of the blood vessel. One potentially important mechanism for endothelial cells to regulate the recruitment of white blood cells is to rapidly modulate their repertoire of cell surface proteins through proteolytic "shedding". In addition to dynamically altering the cell surface constituents, shedding also leads to the release of soluble ectodomains with distinct biological properties. This proposal will focus on the protease ADAM17 that has gained recognition as a key enzyme in proteolytic shedding. We have shown that interference with ADAM17 shedding on endothelial cells is sufficient to inhibit white blood cell migration across cultured endothelial cells, "transendothelial migration". This proposal will further identify and characterize targets of ADAM17-mediated shedding involved in inflammatory responses. We will also examine the steps in transendothelial cell migration that are dependent upon ADAM17-mediated shedding. Finally, we will investigate the impact of in vivo deletion of endothelial cell ADAM17 by evaluating effects on normal endothelial cell functions, including the response to inflammation. The overall goal of our proposal is to test the hypothesis that ADAM17 serves as a gatekeeper during inflammatory responses. The long-term objective of this research is to better understand the cellular and molecular mechanisms controlling chronic inflammatory responses such as atherosclerosis that remains the major cause of death in the Western world.